Human antibodies that bind human tnf-alpha and methods of preparing the same

ABSTRACT

Methylglyoxal (MGO)-modified recombinant TNF-alpha antibodies (e.g., Adalimumab) are identified. MGO modification decreases binding between Adalimumab and TNF-alpha. Methods are disclosed for reducing the presence of MGO-modified antibodies in the production of Adalimumab TNF-alpha antibodies.

RELATED APPLICATION

This application is a division of U.S. patent application Ser. No.14/078,181 filed Nov. 12, 2013, which claims priority to U.S.Provisional Patent Application No. 61/777,883, filed Mar. 12, 2013. Bothof the aforementioned applications are incorporated by reference intothe present application in their entirety and for all purposes.

SEQUENCE LISTING

This application is accompanied by a sequence listing in a computerreadable form that accurately reproduces the sequences described herein.

FIELD OF THE INVENTION

This disclosure relates to antibodies that specifically bind to humanTNF-alpha. More particular, Methylglyoxal (MGO)-modified recombinantTNF-alpha antibodies are disclosed. Methods for reducing MGO-modifiedTNF-alpha antibodies are also provided.

BACKGROUND

Tumor necrosis factor alpha (“TNF-alpha”) is a cytokine produced by manycell types such as monocytes and macrophages. See e.g., Old, L. Science230:630-632 (1985). TNF-alpha plays an important role in many biologicalprocesses and has been implicated in the pathophysiology of a variety ofother human diseases and disorders, including sepsis, infections,autoimmune diseases, transplant rejection and graft-versus-host disease.See e.g., Vasilli, P., Annu. Rev. Immunol. 10:411-452 (1992); andTracey, K. J. and Cerami, A. Annu. Rev. Med. 45:491-503 (1994).

In an effort to treat/prevent these diseases, various therapeuticstrategies have been designed to inhibit or counteract TNF-alphaactivities. U.S. Pat. No. 6,090,382 disclosed human antibodies (e.g.,recombinant human antibodies) that specifically bind to human TNF-alphawith high affinity and slow dissociation kinetics. Nucleic acids,vectors and host cells for expressing the recombinant human TNF-alphaantibodies were also disclosed. One example of such recombinantTNF-alpha antibodies is called Adalimumab, which is marketed under thetrade name Humira®. The entire contents of U.S. Pat. No. 6,090,382 ishereby incorporated by reference into the present disclosure.

Recombinant biotherapeutics are typically produced by live cells and areinherently more complex as compared to traditional small molecule drug.Various post-translational modifications have been reported as majorcontributors to heterogeneity in recombinant monoclonal antibodies(References 1-4). Some of these modifications, for example,glycosylation and sialic acid incorporation, may occur duringfermentation (References 5-7). Some other modifications, such asoxidation and disulfide bond scrambling, may occur during production,purification and storage.

One example of such modifications is the so-called acidic species(charge variants). Acidic species are observed when recombinantmonoclonal antibodies are analyzed by weak-cation exchangechromatography (WCX) (FIG. 1). One major contributing factor is theremoval of the C-terminal lysine of the heavy chain by cell-derivedcarboxypeptidease, reducing the overall positive charge (Reference 8).These variants are commonly referred to as Lys0, Lys1 and Lys2 species,respectively.

C-terminal amidation (Reference 9) is another enzymatic process duringfermentation. Another type of variant is caused by spontaneousnon-enzymatic transformations, which include the formation ofpyruglutamate (Pyro-Glu) from an N-terminal glutamine (Gln) that removethe positive charge of the free N-terminus (Reference 10), and thedeamidation of asparagine (Asn) to aspartic (Asp) or isoaspartic acid(isoAsp or isoD) that introduces negatively charged carboxylic acids(References 11 and 12).

Some modifications may shift the retention time of antibody on weakcation exchange chromatography even though they do not alter the formalcharges of the antibody molecule. These modifications may exert theireffects through perturbation of local charge and conformation. Forinstance, incomplete glycosylation (Reference 13) or the presence offree sulfhydryl (References 14-16) may shift the retention time ofantibody on weak cation exchange chromatography. It is worth noting thatsome modifications are imparted by metabolites, such as glycation byglucose, methionine oxidation by reactive oxygen species (ROS),cysteinylation by cysteine (Reference 17), and S-homocysteinylation andN-homocysteinylation by homocysteine (References 2, 18-23). Although themechanisms of many modifications have been reported, these mechanismscannot fully explained the observed heterogeneity of recombinantmonoclonal antibodies on weak cation exchange chromatography.

SUMMARY

This disclosure advances the art by identifying novel species ofmodified recombinant antibodies that may negatively impact thefunctionalities of such antibodies. The disclosure also provides methodsfor reducing the amount of such species without substantiallycompromising the overall yield of the antibody production.

In one embodiment, two acidic species of the Adalimumab antibody aredisclosed which exist when the antibody are expressed in Chinese hamsterovary (CHO) cells cultured in chemically defined media (CDM). Detailedanalyses have revealed that several arginine residues in Adalimumab aremodified by methylglyoxal (MGO), which is further confirmed by thetreatment of native antibody with authentic MGO. The reaction betweenMGO and arginine result in formation of hydroxylimide and/orhydroimidazolone. The resulting hydroxylimide and hydroimidazoloneadducts increase the molecular weight of the antibody by 54 and 72Daltons, respectively.

In another embodiment, these modifications cause the antibody to eluteearlier in the weak cation exchange chromatogram as compared to theelution time of unmodified forms. Consequently, the extent to which anantibody was modified at multiple sites corresponds to the degree ofshift in acidity and the elution time. The modification of Adalimumabantibody by MGO is the first reported modification of a recombinantmonoclonal antibody by MGO.

In another embodiment, a composition is disclosed which contains abinding protein capable of binding TNF-alpha. In one aspect, the bindingprotein may contain at least one methylglyoxal (MGO)-susceptible aminoacid, and at least a portion of the binding protein may contain one ormore MGO-modified amino acids.

In another embodiment, a composition is disclosed which contains abinding protein capable of binding TNF-alpha. In one aspect, the bindingprotein may contain at least one methylglyoxal (MGO)-susceptible aminoacid and the composition may be prepared by substantially removingmolecules of the binding protein that contain at least one MGO-modifiedamino acid. The term “substantially” may mean at least 50%. In anotheraspect, the term “substantially” may mean at least 60%, 70%, 80%, 90%,or even 100% removal of the molecules that contain at least oneMGO-modified amino acid.

For purpose of this disclosure, the term “methylglyoxal(MGO)-susceptible” refers to groups or residues (e.g., arginine) thatmay react with MGO under appropriate cell culture conditions. List ofMGO-susceptible arginines in Adalimumab is shown in Table 1. Examples ofMGO-susceptible peptides in Adalimumab are shown in Table 2.

The term “at least a portion of the binding protein” means that althoughall molecules of the binding protein in the composition are capable ofbinding TNF-alpha, at least two populations of these molecules exist inthe composition, wherein one population contain one or more amino acidsthat have been modified by MGO, while the other population does notcontain amino acids that have been modified by MGO. In another aspect,all molecules of the binding protein may contain one or more amino acidsthat have been modified by MGO.

In one aspect, the portion of the binding protein that contains at leastone MGO-modified amino acid is less than 15%, 14%, 13%, 12%, 11%, 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the total amount of the bindingprotein.

In another embodiment, the binding protein is a human antibody or anantigen-binding portion thereof, wherein the binding protein dissociatesfrom human TNF-alpha with a K_(d) of 1×10⁻⁸ M or less and a K_(off) rateconstant of 1×10⁻³ s⁻¹ or less, both as determined by surface plasmonresonance. In one aspect, the binding protein neutralizes humanTNF-alpha cytotoxicity in a standard in vitro L929 assay with an IC₅₀ of1×10⁻⁷ M or less, described in Example 4 of U.S. Pat. No. 6,090,382. Inanother aspect, the binding protein is the D2E7 antibody as described inU.S. Pat. No. 6,090,382.

In another embodiment, cell culture parameters may affect the extent ofmodifications by methylglyoxal (MGO). MGO is a highly reactivemetabolite that may be generated from glucose, lipids or other metabolicpathways. In one aspect, cell culture conditions may be modified todecrease the production of MGO thereby reducing modification of therecombinant antibodies by MGO. Taken together, the disclosed findingshighlight the impact of cell culture conditions on the critical qualityattributes of recombinantly produced antibodies. These findings provideadditional parameters for improving manufacturing processes and mayprove useful for the quality by design (QbD) approach.

In another embodiment, methods are disclosed for purifying a targetprotein product from both process and/or product related impurities.Specifically, method for purifying a composition containing a targetprotein is disclosed. In one aspect, methods are provided for reducingproduct related charge variants (i.e. acidic and basic species). Inanother aspect, the method involves contacting the process mixture withan ion (anion or cation) exchange adsorbent in an aqueous salt solutionunder loading conditions that permit both the target and non-targetproteins to bind to the adsorbent and allowing the excess targetmolecule to pass through the column and subsequently recovering thebound target protein with a wash at the same aqueous salt solution usedin the equilibration (i.e. pre-loading) condition.

In another embodiment, a method for purifying a composition containing atarget protein is disclosed which may include at least the followingsteps: (a) loading the composition to a cation exchange adsorbent usinga loading buffer, wherein the pH of the loading buffer is lower than thepI of the target protein; (b) washing the cation exchange adsorbent witha washing buffer, wherein the pH of the washing buffer is lower than thepI of the target protein; (c) eluting the cation exchange adsorbent withan elution buffer, said elution buffer being capable of reducing thebinding between the target protein and the cation exchange adsorbent;and (d) collecting the eluate, wherein the percentage of the targetprotein is higher in the eluate than the percentage of the targetprotein in the composition. In one aspect, the washer buffer and theloading buffer are the same. In another aspect, the conductivity of theelution buffer is higher than the conductivity of the washer buffer. Inanother aspect, the pH of the elution buffer may be between 5.5 and 9.0,between 6 and 8, or between 6.5 and 8. The conductivity of the elutionbuffer may be raised by increasing the salt concentration of the elutionbuffer. The salt concentration of the elution buffer may be between 20mM NaCl and 200 mM NaCl, between 40 mM NaCl and 160 mM NaCl, or between60 mM NaCl and 120 mM NaCl.

In another embodiment, a method for purifying a composition containing atarget protein is disclosed which may include at least the followingsteps: (a) loading the composition to an anion exchange adsorbent usinga loading buffer, wherein the pH of the loading buffer is lower than theisoelectric point (pI) of the target protein; (b) allowing the majorityof the target protein to pass through without binding to the anionexchange adsorbent; (c) collecting the pass-through loading buffercontaining said unbound target protein; (d) washing the anion exchangeadsorbent with a washing buffer; (e) allowing the target protein boundto the anion exchange adsorbent to disassociate from the anion exchangeadsorbent; (f) collecting the washing buffer containing saiddisassociated target protein. In another aspect, the method may furtherinclude a step (g) of pooling the collections from steps (c) and (f) toobtain a purified composition containing the target protein. Thepercentage of the target protein is higher in the pooled collectionsthan the percentage of the target protein in the original composition.

In one aspect, the loading buffer may contain an anionic agent and acationic agent, wherein the conductivity and pH of the loading buffer isadjusted by increasing or decreasing the concentration of a cationicagent and maintaining a constant concentration of an anionic agent inthe loading buffer. In another aspect, the anionic agent is selectedfrom the group consisting of acetate, citrate, chloride anion, sulphate,phosphate and combinations thereof. In another aspect, the cationicagent is selected from the group consisting of sodium, Tris,tromethalmine, ammonium cation, arginine, and combinations thereof.

In one embodiment, the target protein is a human antibody or anantigen-binding portion thereof that is substantially free from MGOmodification. In one aspect, the target protein dissociates from humanTNF-alpha with a K_(d) of 1×10⁻⁸ M or less and a K_(off) rate constantof 1×10⁻³ s⁻¹ or less, both as determined by surface plasmon resonance.In another aspect, the target protein neutralizes human TNF-alphacytotoxicity in a standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁷ Mor less, described in Example 4 of U.S. Pat. No. 6,090,382. In anotheraspect, the target protein is the D2E7 antibody as described in U.S.Pat. No. 6,090,382.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a typical WCX chromatogram of adalimumab after protein Apurification.

FIG. 2 shows deconvoluted mass spectra of the light chain and heavychains in fractions 1 and 2.

FIG. 3 shows representative MS/MS mass spectra of peptides containingArg residues modified by MGO.

FIG. 4 shows chemical modification of arginine by MGO.

FIG. 5 shows modification of a purified 0 lysine fraction by MGO over a5-hour time course.

FIG. 6 shows the mass spectra of peaks a and b from FIG. 5.

FIG. 7 shows comparison of peptide MS/MS data between acidic fraction 1from cell culture and acidic fraction 1 from methylglyoxal incubation.

FIG. 8 shows the crystal structure of the adalimumab Fab subunit incomplex with TNF-alpha, indicating that modification by MGO may causeconformational change which may impede adalimumab's ability to bindTNF-alpha.

FIG. 9 shows Surface Plasmon Resonance (SPR) data for 0 Lys Fraction(Top—0 Lys) and for the MGO enriched fraction (Bottom—Peak 1).

FIG. 10 shows comparison of acidic region affected by methylglyoxalbefore and after two-step chromatographic separation, wherein the toptrace is an expanded view of the acidic region in which the twodistinctive MGO peaks are denoted, and the lower trace shows a completeclearance of this acidic region and the MGO variants.

FIG. 11 shows the CEX chromatogram when reversible binding mode wasperformed using Adalimumab with a Tris-acetate buffer system.

FIG. 12 shows the removal of acidic species by Poros XS resin withNaCl/Tris-acetate solution.

DETAILED DESCRIPTION

The instant disclosure identifies novel species of methylglyoxal(MGO)-modified recombinant antibodies which may have negative impact onthe structure and function of the antibodies. The disclosure alsoprovides methods for reducing the percentage of such variant specieswithout substantially compromising the yield of antibody production.More specifically, this disclosure describes methylglyoxal(MGO)-modified forms of Adalimumab in cell culture when Adalimumab isexpressed in CHO cells using chemically defined media (CDM).

In one embodiment, modification of the side chain of certain arginines(e.g., R30 in CDR1 of Adalimumab) by MGO may result in the formation ofa five-member ring originating at the guanidinium terminal of the sidechain which may further penetrate into the TNF-alpha structure. TheseMGO modifications may impede Adalimumab's ability to bind TNF-alpha dueto steric constraints.

In one embodiment, control of acidic species heterogeneity may beattained by purifying a protein of interest from a mixture comprisingthe protein with an anion exchange (AEX) adsorbent material and anaqueous salt solution under loading conditions that permit both theprotein of interest and non-target proteins to bind to the AEXadsorbent, wherein the bound protein of interest is subsequentlyrecovered with a wash buffer comprising the same aqueous salt solutionused in the equilibration (i.e. loading) buffer. In one aspect, theaqueous salt solution used as both the loading and wash buffer has a pHthat is greater than the isoelectric point (pI) of the protein ofinterest.

In another embodiment, the disclosed purification method may includeadjusting the conductivity and/or pH of the aqueous salt solution. Inone aspect, the adjustments may include decreasing the conductivity ofthe aqueous salt solution. In another aspect, the adjustment to achievethe desired control over acidic species heterogeneity may involve anincrease in the load conductivity of the solution. In another aspect,the adjustment may increase the pH of the aqueous salt solution. Inanother aspect, the adjustment to achieve the desired control overacidic species heterogeneity may involve a decrease in the pH of theaqueous salt solution. Such increases and/or decreases in theconductivity and/or pH may be of a magnitude of 1%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100%, and ranges within one or more of the preceding, of theconductivity and/or pH of the aqueous salt solution.

In another embodiment, the conductivity and pH of the aqueous saltsolution is adjusted by increasing or decreasing the concentration of acationic agent and maintaining a constant concentration of an anionicagent in the aqueous salt solution. In one aspect, the anionic agent ismaintained at a concentration of between about 0.05 mM and 100 mM, orbetween about 0.1 mM and 90 mM, or between about 0.5 mM and 80 mM, orbetween about 1 mM and 70 mM, or between about 1.5 mM and 60 mM, orbetween about 2 mM and 50 mM, or between about 2.5 mM and 40 mM, orbetween about 3 mM and 30 mM, or between about 3.5 mM and 25 mM, orbetween about 4 mM and 20 mM, or between about 4.5 mM and 15 mM, orbetween about 4.5 mM and 10 mM, or between about 5 mM and 7 mM. Inanother aspect, the anionic agent is maintained at a concentration ofabout 5 mM. In another aspect, the anionic agent is maintained at aconcentration of about 10 mM. In another aspect, the anionic agent ismaintained at a concentration of about 18.5 mM.

In another embodiment, the concentration of the cationic agent in theaqueous salt solution is increased or decreased to achieve a pH ofbetween about 5 and 12, or between about 5.5 and 11.5, or between about6 and 11, or between about 6.5 and 10.5, or between about 7 and 10, orbetween about 7.5 and 9.5, or between about 8 and 9, or between about8.5 and 9. In certain embodiments, the concentration of cationic agentis increased or decreased in the aqueous salt solution to achieve a pHof 8.8. In certain embodiments, the concentration of cationic agent inthe aqueous salt solution is increased or decreased to achieve a pH of9.

In another embodiment, the protein load of the protein mixture isadjusted to a protein load of between about 50 g/L and 500 g/L, orbetween about 100 g/L and 450 g/L, or between about 120 g/L and 400 g/L,or between about 125 g/L and 350 g/L, or between about 130 g/L and 300g/L or between about 135 g/L and 250 g/L, or between about 140 g/L and200 g/L, or between about 145 g/L and 200 g/L, or between about 150 g/Land 200 g/L, or between about 155 g/L and 200 g/L, or between about 160g/L and 200 g/L. In certain embodiments, the protein load of the proteinor antibody mixture is adjusted to a protein load of about 100 g/L. Incertain embodiments, the protein load of the protein or antibody mixtureis adjusted to a protein load of about 20 g/L. In certain embodiments,the protein load of the protein or antibody mixture is adjusted to aprotein load of about 105 g/L. In certain embodiments, the protein loadof the protein or antibody mixture is adjusted to a protein load ofabout 140 g/L. In certain embodiments, the protein load of the proteinor antibody mixture is adjusted to a protein load of about 260 g/L. Incertain embodiments, the protein load of the protein or antibody mixtureis adjusted to a protein load of about 300 g/L.

In another embodiment, the concentration of cationic agent in theaqueous salt solution is increased or decreased in an amount effectiveto reduce the amount of acidic species heterogeneity in a protein orantibody sample by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%,4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, and ranges within oneor more of the preceding, when the aqueous salt solution is used as aload and wash buffer to purify the protein of interest (for example, anantibody) from the sample containing the protein.

In another embodiment, the anionic agent is acetate, citrate, chlorideanion, sulphate, phosphate or combinations thereof. In certainembodiments, the cationic agent is sodium, Tris, tromethalmine, ammoniumcation, arginine, or combinations thereof.

By way of example but not limitation, as detailed in this disclosure, upto 60% of the acidic species in an antibody preparation was removed whenthe antibody was purified using chromatography comprising an anionexchange adsorbent material, a protein load of 150 g/L, and a load/washbuffer containing 5 mM Acetate/Arginine at pH 8.8.

In another embodiment of the instant disclosure, control of acidicspecies heterogeneity can be attained by purifying a protein of interestfrom a mixture comprising the protein with a cation exchange (CEX)adsorbent material and an aqueous salt solution under loading conditionsthat permit both the protein of interest and non-target proteins to bindto the CEX adsorbent, washing off the acidic species, charged variants,molecular variants and impurities using the same buffer conditions asthe loading buffer, and eluting the bound protein target from the CEXadsorbent with a buffer having a higher conductivity than the loadingbuffer. In certain embodiments, the aqueous salt solution used as boththe loading and wash buffer has a pH that is lower than the isoelectricpoint (pI) of the protein of interest.

In another embodiment, the purification method may include adjusting theconductivity and/or pH of the aqueous solution. In certain embodiments,such adjustments will be to decrease the conductivity, while in otherembodiments the necessary adjustment to achieve the desired control overacidic species heterogeneity will involve an increase in the loadconductivity. In certain embodiments, such adjustments will also be toincrease the pH of the aqueous salt solution, while in other embodimentsthe necessary adjustment to achieve the desired control over acidicspecies heterogeneity will involve a decrease in the pH of the aqueoussalt solution. Such increases and/or decreases in the conductivityand/or pH can be of a magnitude of 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, andranges within one or more of the preceding, of the conductivity and/orpH of the aqueous salt solution.

In certain embodiments, the conductivity and pH of the aqueous saltsolution is adjusted by increasing or decreasing the concentration of aanionic agent and maintaining a constant concentration of a cationicagent in the aqueous salt solution. In certain embodiments, the cationicagent is maintained at a concentration of between about 0.5 mM and 500mM, or between about 1 mM and 450 mM, or between about 5 mM and 400 mM,or between about 10 mM and 350 mM, or between about 15 mM and 300 mM, orbetween about 20 mM and 250 mM, or between about 25 mM and 200 mM, orbetween about 30 mM and 150 mM, or between about 35 mM and 100 mM, orbetween about 40 mM and 50 mM. In certain embodiments, the anionic agentis maintained at a concentration of about 15 mM, or about 20 mM, orabout 25 mM, or about 30 mM, or about 35 mM, or about 40 mM, or about 45mM, or about 50 mM, or about 60 mM, or about 65 mM, or about 75 mM, orabout 90 mM, or about 115 mM, or about 120 mM, or about 125 mM, or about135 mM, or about 140 mM, or about 145 mM, or about 150 mM, or about 175mM, or about 250 mM, or about 275 mM, or about 300 mM, or about 350 mM,or about 375 mM, or about 400 mM.

In certain embodiments, the concentration of the anionic agent inaqueous salt solution is increased or decreased to achieve a pH ofbetween about 2 and 12, or between about 2.5 and 11.5, or between about3 and 11, or between about 3.5 and 10.5, or between about 4 and 10, orbetween about 4.5 and 9.5, or between about 5 and 9, or between about5.5 and 8.5, or between about 6 and 8, or between about 6.5 and 7.5. Incertain embodiments, the concentration of anionic agent is increased ordecreased in the aqueous salt solution to achieve a pH of 5, or 5.5, or6, or 6.5, or 6.8, or 7.5.

In certain embodiments, the protein load of the protein mixture isadjusted to a protein load of between about 50 and 500 g/L, or betweenabout 100 and 450 g/L, or between about 120 and 400 g/L, or betweenabout 125 and 350 g/L, or between about 130 and 300 g/L or between about135 and 250 g/L, or between about 140 and 200 g/L, or between about 145and 150 g/L. In certain embodiments, the protein load of the protein orantibody mixture is adjusted to a protein load of about 40 g/L.

In certain embodiments, the concentration of anionic agent in theaqueous salt solution is increased or decreased in an amount effectiveto reduce the amount of acidic species heterogeneity in a protein orantibody sample by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%,4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, and ranges within oneor more of the preceding, when the aqueous salt solution is used as aload and wash buffer to purify the protein of interest (for example, anantibody) from the sample containing the protein.

In certain embodiments, the cationic agent is sodium, Tris,tromethalmine, ammonium cation, arginine, or combinations thereof. Incertain embodiments, the anionic agent is acetate, citrate, chlorideanion, sulphate, phosphate or combinations thereof.

By way of example but not limitation, as detailed in this disclosure,the presence of acidic species in an antibody preparation was reduced by6.5% from starting material after purification using a cation exchangeadsorbent material, and a load and wash buffer comprising 140 mM Tris atpH 7.5.

Unless otherwise defined herein, scientific and technical terms usedherein have the meanings that are commonly understood by those ofordinary skill in the art. In the event of any latent ambiguity,definitions provided herein take precedent over any dictionary orextrinsic definition. Unless otherwise required by context, singularterms shall include pluralities and plural terms shall include thesingular. The use of “or” means “and/or” unless stated otherwise. Theuse of the term “including”, as well as other forms, such as “includes”and “included”, is not limiting.

Generally, nomenclatures used in connection with cell and tissueculture, molecular biology, immunology, microbiology, genetics andprotein and nucleic acid chemistry and hybridization described hereinare those well known and commonly used in the art. The methods andtechniques provided herein are generally performed according toconventional methods well known in the art and as described in variousgeneral and more specific references that are cited and discussedthroughout the present specification unless otherwise indicated.Enzymatic reactions and purification techniques are performed accordingto manufacturer's specifications, as commonly accomplished in the art oras described herein. The nomenclatures used in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those well known and commonly used in the art. Standardtechniques are used for chemical syntheses, chemical analyses,pharmaceutical preparation, formulation, and delivery, and treatment ofpatients.

That the disclosure may be more readily understood, select terms aredefined below.

The term “antibody” refers to an immunoglobulin (Ig) molecule, which isgenerally comprised of four polypeptide chains, two heavy (H) chains andtwo light (L) chains, or a functional fragment, mutant, variant, orderivative thereof, that retains the epitope binding features of an Igmolecule. Such fragment, mutant, variant, or derivative antibody formatsare known in the art. In an embodiment of a full-length antibody, eachheavy chain is comprised of a heavy chain variable region (VH) and aheavy chain constant region (CH). The heavy chain variable region(domain) is also designated as VDH in this disclosure. The CH iscomprised of three domains, CH1, CH2 and CH3. Each light chain iscomprised of a light chain variable region (VL) and a light chainconstant region (CL). The CL is comprised of a single CL domain. Thelight chain variable region (domain) is also designated as VDL in thisdisclosure. The VH and VL can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDRs),interspersed with regions that are more conserved, termed frameworkregions (FRs). Generally, each VH and VL is composed of three CDRs andfour FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD,IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), orsubclass.

The term “antigen-binding portion” of an antibody (or “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., hTNF-alpha). It has been shown that the antigen-binding functionof an antibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH I domains; (ii)a F(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426: and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.Other forms of single chain antibodies, such as diabodies are alsoencompassed. Diabodies are bivalent, bispecific antibodies in which VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell, antibodiesisolated from a recombinant, combinatorial human antibody library,antibodies isolated from an animal (e.g., a mouse) that is transgenicfor human immunoglobulin genes (see e.g., Taylor, L. D., et al. (1992)Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed,created or isolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the VH and VLregions of the recombinant antibodies are sequences that, while derivedfrom and related to human germline VH and VL sequences, may notnaturally exist within the human antibody germline repertoire in vivo.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in proteinconcentrations within a biosensor matrix, for example using the BIAcoresystem (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).For further descriptions, see Example 1 and Jonsson, U., et al. (1993)Ann. Biol. Clin. 51:19-26; Jonsson, U., et al. (1991) Biotechniques11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; andJohnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

The term “biological activity” refers to any one or more biologicalproperties of a molecule (whether present naturally as found in vivo, orprovided or enabled by recombinant means). Biological propertiesinclude, but are not limited to, binding a receptor or receptor ligand,inducing cell proliferation, inhibiting cell growth, inducing othercytokines, inducing apoptosis, and enzymatic activity.

The term “neutralizing” refers to counteracting the biological activityof an antigen/ligand when a binding protein specifically binds to theantigen/ligand. In an embodiment, the neutralizing binding protein bindsto an antigen/ligand (e.g., a cytokine) and reduces its biologicallyactivity by at least about 20%, 40%, 60%, 80%, 85% or more.

“Specificity” refers to the ability of a binding protein to selectivelybind an antigen/ligand.

“Affinity” is the strength of the interaction between a binding proteinand an antigen/ligand, and is determined by the sequence of the bindingdomain(s) of the binding protein as well as by the nature of theantigen/ligand, such as its size, shape, and/or charge. Binding proteinsmay be selected for affinities that provide desired therapeuticend-points while minimizing negative side-effects. Affinity may bemeasured using methods known to one skilled in the art (US 20090311253).

The term “potency” refers to the ability of a binding protein to achievea desired effect, and is a measurement of its therapeutic efficacy.Potency may be assessed using methods known to one skilled in the art(US 20090311253).

The term “cross-reactivity” refers to the ability of a binding proteinto bind a target other than that against which it was raised. Generally,a binding protein will bind its target tissue(s)/antigen(s) with anappropriately high affinity, but will display an appropriately lowaffinity for non-target normal tissues. Individual binding proteins aregenerally selected to meet two criteria. (1) Tissue staining appropriatefor the known expression of the antibody target. (2) Similar stainingpattern between human and tox species (mouse and cynomolgus monkey)tissues from the same organ. These and other methods of assessingcross-reactivity are known to one skilled in the art (US 20090311253).

The term “biological function” refers the specific in vitro or in vivoactions of a binding protein. Binding proteins may target severalclasses of antigens/ligands and achieve desired therapeutic outcomesthrough multiple mechanisms of action. Binding proteins may targetsoluble proteins, cell surface antigens, as well as extracellularprotein deposits. Binding proteins may agonize, antagonize, orneutralize the activity of their targets. Binding proteins may assist inthe clearance of the targets to which they bind, or may result incytotoxicity when bound to cells. Portions of two or more antibodies maybe incorporated into a multivalent format to achieve distinct functionsin a single binding protein molecule. The in vitro assays and in vivomodels used to assess biological function are known to one skilled inthe art (US 20090311253).

The term “solubility” refers to the ability of a protein to remaindispersed within an aqueous solution. The solubility of a protein in anaqueous formulation depends upon the proper distribution of hydrophobicand hydrophilic amino acid residues, and therefore, solubility cancorrelate with the production of correctly folded proteins. A personskilled in the art will be able to detect an increase or decrease insolubility of a binding protein using routine HPLC techniques andmethods known to one skilled in the art (US 20090311253).

Binding proteins may be produced using a variety of host cells or may beproduced in vitro, and the relative yield per effort determines the“production efficiency.” Factors influencing production efficiencyinclude, but are not limited to, host cell type (prokaryotic oreukaryotic), choice of expression vector, choice of nucleotide sequence,and methods employed. The materials and methods used in binding proteinproduction, as well as the measurement of production efficiency, areknown to one skilled in the art (US 20090311253).

The term “conjugate” refers to a binding protein, such as an antibody,that is chemically linked to a second chemical moiety, such as atherapeutic or cytotoxic agent. The term “agent” includes a chemicalcompound, a mixture of chemical compounds, a biological macromolecule,or an extract made from biological materials. In an embodiment, thetherapeutic or cytotoxic agents include, but are not limited to,pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide,emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine,colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione,mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. When employed in the contextof an immunoassay, the conjugate antibody may be a detectably labeledantibody used as the detection antibody.

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double stranded DNAloop into which additional DNA segments may be ligated. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Other vectors include RNA vectors. Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., bacterial vectors having a bacterial originof replication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “recombinant expression vectors”(or simply, “expression vectors”). In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification, “plasmid” and “vector” may be usedinterchangeably as the plasmid is the most commonly used form of vector.However, other forms of expression vectors are also included, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions. A groupof pHybE vectors (U.S. Patent Application Ser. No. 61/021,282) were usedfor parental binding protein cloning.

The terms “recombinant host cell” or “host cell” refer to a cell intowhich exogenous DNA has been introduced. Such terms refer not only tothe particular subject cell, but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein. In an embodiment, host cellsinclude prokaryotic and eukaryotic cells. In an embodiment, eukaryoticcells include protist, fungal, plant and animal cells. In anotherembodiment, host cells include but are not limited to the prokaryoticcell line E. Coli; mammalian cell lines CHO, HEK293, COS, NS0, SP2 andPER.C6; the insect cell line Sf9; and the fungal cell Saccharomycescerevisiae.

The term “transfection” encompasses a variety of techniques commonlyused for the introduction of exogenous nucleic acid (e.g., DNA) into ahost cell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like.

The term “cytokine” refers to a protein released by one cell populationthat acts on another cell population as an intercellular mediator. Theterm “cytokine” includes proteins from natural sources or fromrecombinant cell culture and biologically active equivalents of thenative sequence cytokines.

The term “biological sample” means a quantity of a substance from aliving thing or formerly living thing. Such substances include, but arenot limited to, blood, (e.g., whole blood), plasma, serum, urine,amniotic fluid, synovial fluid, endothelial cells, leukocytes,monocytes, other cells, organs, tissues, bone marrow, lymph nodes andspleen.

The term “component” refers to an element of a composition. In relationto a diagnostic kit, for example, a component may be a capture antibody,a detection or conjugate antibody, a control, a calibrator, a series ofcalibrators, a sensitivity panel, a container, a buffer, a diluent, asalt, an enzyme, a co-factor for an enzyme, a detection reagent, apretreatment reagent/solution, a substrate (e.g., as a solution), a stopsolution, and the like that can be included in a kit for assay of a testsample. Thus, a “component” can include a polypeptide or other analyteas above, that is immobilized on a solid support, such as by binding toan anti-analyte (e.g., anti-polypeptide) antibody. Some components canbe in solution or lyophilized for reconstitution for use in an assay.

“Control” refers to a composition known to not analyte (“negativecontrol”) or to contain analyte (“positive control”). A positive controlcan comprise a known concentration of analyte. “Control,” “positivecontrol,” and “calibrator” may be used interchangeably herein to referto a composition comprising a known concentration of analyte. A“positive control” can be used to establish assay performancecharacteristics and is a useful indicator of the integrity of reagents(e.g., analytes).

The term “Fc region” defines the C-terminal region of an immunoglobulinheavy chain, which may be generated by papain digestion of an intactantibody. The Fc region may be a native sequence Fc region or a variantFc region. The Fc region of an immunoglobulin generally comprises twoconstant domains, a CH2 domain and a CH3 domain, and optionallycomprises a CH4 domain. Replacements of amino acid residues in the Fcportion to alter antibody effector function are known in the art (e.g.,U.S. Pat. Nos. 5,648,260 and 5,624,821). The Fc region mediates severalimportant effector functions, e.g., cytokine induction, antibodydependent cell mediated cytotoxicity (ADCC), phagocytosis, complementdependent cytotoxicity (CDC), and half-life/clearance rate of antibodyand antigen-antibody complexes. In some cases these effector functionsare desirable for a therapeutic immunoglobulin but in other cases mightbe unnecessary or even deleterious, depending on the therapeuticobjectives.

The terms “Kabat numbering”, “Kabat definitions” and “Kabat labeling”are used interchangeably herein. These terms, which are recognized inthe art, refer to a system of numbering amino acid residues which aremore variable (i.e., hypervariable) than other amino acid residues inthe heavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al. (1971) Ann. NY Acad. Sci.190:382-391 and, Kabat et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). For the heavy chainvariable region, the hypervariable region ranges from amino acidpositions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, andamino acid positions 95 to 102 for CDR3. For the light chain variableregion, the hypervariable region ranges from amino acid positions 24 to34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acidpositions 89 to 97 for CDR3.

The term “CDR” means a complementarity determining region within animmunoglobulin variable region sequence. There are three CDRs in each ofthe variable regions of the heavy chain and the light chain, which aredesignated CDR1, CDR2 and CDR3, for each of the heavy and light chainvariable regions. The term “CDR set” refers to a group of three CDRsthat occur in a single variable region capable of binding the antigen.The exact boundaries of these CDRs have been defined differentlyaccording to different systems. The system described by Kabat (Kabat etal. (1987) and (1991)) not only provides an unambiguous residuenumbering system applicable to any variable region of an antibody, butalso provides precise residue boundaries defining the three CDRs. TheseCDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothiaand Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature342:877-883) found that certain sub-portions within Kabat CDRs adoptnearly identical peptide backbone conformations, despite having greatdiversity at the level of amino acid sequence. These sub-portions weredesignated as L1, L2 and L3 or H1, H2 and H3 where the “L” and the “H”designates the light chain and the heavy chain regions, respectively.These regions may be referred to as Chothia CDRs, which have boundariesthat overlap with Kabat CDRs. Other boundaries defining CDRs overlappingwith the Kabat CDRs have been described by Padlan (1995) FASEB J.9:133-139 and MacCallum (1996) J. Mol. Biol. 262(5):732-45). Still otherCDR boundary definitions may not strictly follow one of the hereinsystems, but will nonetheless overlap with the Kabat CDRs, although theymay be shortened or lengthened in light of prediction or experimentalfindings that particular residues or groups of residues or even entireCDRs do not significantly impact antigen binding. The methods usedherein may utilise CDRs defined according to any of these systems,although certain embodiments use Kabat or Chothia defined CDRs.

The term “epitope” means a region of an antigen that is bound by abinding protein, e.g., a polypeptide and/or other determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. In certainembodiments, epitope determinants include chemically active surfacegroupings of molecules such as amino acids, sugar side chains,phosphoryl, or sulfonyl, and, in certain embodiments, may have specificthree dimensional structural characteristics, and/or specific chargecharacteristics. In an embodiment, an epitope comprises the amino acidresidues of a region of an antigen (or fragment thereof) known to bindto the complementary site on the specific binding partner. An antigenicfragment can contain more than one epitope. In certain embodiments, abinding protein specifically binds an antigen when it recognizes itstarget antigen in a complex mixture of proteins and/or macromolecules.Binding proteins “bind to the same epitope” if the antibodiescross-compete (one prevents the binding or modulating effect of theother). In addition, structural definitions of epitopes (overlapping,similar, identical) are informative; and functional definitionsencompass structural (binding) and functional (modulation, competition)parameters. Different regions of proteins may perform differentfunctions. For example specific regions of a cytokine interact with itscytokine receptor to bring about receptor activation whereas otherregions of the protein may be required for stabilizing the cytokine. Toabrogate the negative effects of cytokine signaling, the cytokine may betargeted with a binding protein that binds specifically to the receptorinteracting region(s), thereby preventing the binding of its receptor.Alternatively, a binding protein may target the regions responsible forcytokine stabilisation, thereby designating the protein for degradation.The methods of visualizing and modeling epitope recognition are known toone skilled in the art (US 20090311253).

“Pharmacokinetics” refers to the process by which a drug is absorbed,distributed, metabolized, and excreted by an organism. To generate amultivalent binding protein molecule with a desired pharmacokineticprofile, parent binding proteins with similarly desired pharmacokineticprofiles are selected. The PK profiles of the selected parental bindingproteins can be easily determined in rodents using methods known to oneskilled in the art (US 20090311253).

“Bioavailability” refers to the amount of active drug that reaches itstarget following administration. Bioavailability is function of severalof the previously described properties, including stability, solubility,immunogenicity and pharmacokinetics, and can be assessed using methodsknown to one skilled in the art (US 20090311253).

The term “K_(on)” means the on rate constant for association of abinding protein (e.g., an antibody) to the antigen to form the,antibody/antigen complex. The term “K_(on)” also means “association rateconstant”, or “ka”, as is used interchangeably herein. This valueindicating the binding rate of a binding protein to its target antigenor the rate of complex formation between a binding protein, e.g., anantibody, and antigen also is shown by the equation below:

Antibody (“Ab”)+Antigen (“Ag”)→Ab-Ag

The term “K_(off)” means the off rate constant for dissociation, or“dissociation rate constant”, of a binding protein (e.g., an antibody)from the, antibody/antigen complex as is known in the art. This valueindicates the dissociation rate of a binding protein, e.g., an antibody,from its target antigen or separation of Ab-Ag complex over time intofree antibody and antigen as shown by the equation below:

Ab+Ag←Ab-Ag

The terms “K_(d)” and “equilibrium dissociation constant” means thevalue obtained in a titration measurement at equilibrium, or by dividingthe dissociation rate constant (K_(off)) by the association rateconstant (K_(on)). The association rate constant, the dissociation rateconstant and the equilibrium dissociation constant, are used torepresent the binding affinity of a binding protein (e.g., an antibody)to an antigen. Methods for determining association and dissociation rateconstants are well known in the art. Using fluorescence based techniquesoffers high sensitivity and the ability to examine samples inphysiological buffers at equilibrium. Other experimental approaches andinstruments such as a BIAcore® (biomolecular interaction analysis)assay, can be used (e.g., instrument available from BIAcoreInternational AB, a GE Healthcare company, Uppsala, Sweden).Additionally, a KinExA® (Kinetic Exclusion Assay) assay, available fromSapidyne Instruments (Boise, Id.), can also be used.

The term “variant” means a polypeptide that differs from a givenpolypeptide in amino acid sequence or in post-translationalmodification. The difference in amino acid sequence may be caused by theaddition (e.g., insertion), deletion, or conservative substitution ofamino acids, but that retains the biological activity of the givenpolypeptide (e.g., a variant TNF-alpha antibody can compete withanti-TNF-alpha antibody for binding to TNF-alpha). A conservativesubstitution of an amino acid, i.e., replacing an amino acid with adifferent amino acid of similar properties (e.g., hydrophilicity anddegree and distribution of charged regions) is recognized in the art astypically involving a minor change. These minor changes can beidentified, in part, by considering the hydropathic index of aminoacids, as understood in the art (see, e.g., Kyte et al. (1982) J. Mol.Biol. 157: 105-132). The hydropathic index of an amino acid is based ona consideration of its hydrophobicity and charge. It is known in the artthat amino acids of similar hydropathic indexes in a protein can besubstituted and the protein still retains protein function. In oneaspect, amino acids having hydropathic indexes of ±2 are substituted.The hydrophilicity of amino acids also can be used to revealsubstitutions that would result in proteins retaining biologicalfunction. A consideration of the hydrophilicity of amino acids in thecontext of a peptide permits calculation of the greatest local averagehydrophilicity of that peptide, a useful measure that has been reportedto correlate well with antigenicity and immunogenicity (see, e.g., U.S.Pat. No. 4,554,101). Substitution of amino acids having similarhydrophilicity values can result in peptides retaining biologicalactivity, for example immunogenicity, as is understood in the art. Inone aspect, substitutions are performed with amino acids havinghydrophilicity values within ±2 of each other. Both the hydrophobicityindex and the hydrophilicity value of amino acids are influenced by theparticular side chain of that amino acid. Consistent with thatobservation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties. The term “variant” also includes polypeptide orfragment thereof that has been differentially processed, such as byproteolysis, phosphorylation, or other post-translational modification,yet retains its biological activity or antigen reactivity, e.g., theability to bind to TNF-alpha. The term “variant” encompasses fragmentsof a variant unless otherwise defined. A variant may be 99%, 98%, 97%,96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%,82%, 81%, 80%, 79%, 78%, 77%, 76%, or 75% identical to the wild-typesequence.

The difference in post-translational modification may be effected byaddition of one or more chemical groups to the amino acids of themodified molecule, or removal of one or more such groups from themolecule. Examples of modification may include but are not limited to,phosphorylation, glysosylation, or MGO modification.

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods described hereinare obvious and may be made using suitable equivalents without departingfrom the scope of the embodiments disclosed herein. Having now describedcertain embodiments in detail, the same will be more clearly understoodby reference to the following examples, which are included for purposesof illustration only and are not intended to be limiting.

EXAMPLES Example 1 Identification of Different Forms of MGO-mAb

In a traditional process for making Adalimumab, antibody expressiontypically takes place by using Hydrolysate and Phytone as raw materials.When adalimumab was expressed with CHO cells using chemically definedmedia (CDM), the percentage of acidic species as defined by the weakcation exchange chromatography method increased as compared to thepercentage of acidic species generated by the traditional productionprocess. Specifically, two distinct early eluting chromatographic peakswere observed as shown in FIG. 1. The peaks labeled as Lys 0, Lys 1 andLys 2 are antibody without C-terminal Lys, with one C-terminal Lys andwith two C-terminal Lys on the heavy chains, respectively. The top traceis from adalimumab produced using chemically defined media (CDM) and thebottom trace is from adalimumab produced using yeastolate. Two peakswere observed in antibody expressed in cell culture using CDM and aredenoted by Fractions 1 and 2, respectively. These peaks are unique toadalimumab production with CDM. The peaks were subsequently isolatedusing weak cation exchange fractionation.

Analysis of the isolated peaks by reduced LC/MS revealed mass spectra ofthe expected values for the adalimumab heavy chain and light chain butwith additional peak corresponding to mass increases of +54 Da and +72Da with additional lower intensity peaks which are likely due toadditional modifications at multiple sites of the respective chains(FIG. 2). As shown in FIG. 2 left panel, three major peaks correspondingto the theoretical molecular weight of the light chain at 23408 Da plusmasses of 23462 and 23480 were observed. The two peaks that shift fromthe theoretical molecular weight diverge from the expected mass byincreases of 54 and 72 daltons, respectively. As shown in FIG. 2 RightPanel, three peaks corresponding to the theoretical molecular weight ofthe heavy chain at 50637 Da plus an additional ladder of massescorresponding to 54 and 72 Da increases were observed. Peaks with thesemolecular weight increases were observed for both the light chain andheavy chain from fractions 1 and 2 but were noticeably absent from theLys-0 controls (bottom spectra of FIG. 2).

The peaks were subsequently analyzed by peptide mapping with LC/MS/MSdetection. Modifications that resulted in the molecular weight increasesof both 54 Da and 72 Da were localized to a particular Arg for thispeptide and has resulted in a tryptic mis-cleavage (FIG. 3). Thisobservation supports the hypothesis of hydroxylimidine conversion to ahydroimadazolone after loss of water. The results suggest that themodifications are localized to miscleaved tryptic peptides where theadduction is on the arginine side chain.

Based on these observations, it is likely that the adduction of theantibody was due to methylglyoxal (MGO) accumulation in cell culturesgrown in the presence of chemically defined media (CDM). The reactionscheme for methylglyoxal modification of arginine residues is shown inFIG. 4. The initial adduction of MGO with an arginine side chain resultsin the formation of a hydroxylimidine with an observed mass increase of+72Da. Following a dehydration to a hydroimadazolone, the resultingproduct has a +54 Da mass increase.

In order to confirm that an accumulation of methylglyoxal is the causeof the +54 Da and +72 Da mass increases associated with the earlyeluting acidic peaks, antibody was incubated with syntheticmethylglyoxal and analyzed over a time course. WCX-10 fractionation wasused to isolate zero lysine species, which is the adalimumab antibodywith only the dominant main peak of the weak cation exchangechromatogram present. The 0 Lys species was incubated in the presence of2.7 mM MGO over the course of five hours at 37 C.

As shown in FIG. 5, over the time course, nearly all of the 0 Lys wasconverted to the two distinct acidic peaks found in the initial materialanalyzed from the CDM expressions. The lysine 0 after incubation underthe same condition without exposure to MGO is also shown as a control.Peaks a and b from the sample treated with MGO for 120 minutes weresubsequently collected and analyzed by LC/MS to assess the level ofchemical modifications which have resulted.

Subsequent analysis of 0 Lys material incubated with MGO showed thepreviously observed ladder of +54 Da and +72 Da mass heterogeneity as aprevalent pattern in the mass spectra of both the adalimumab light chainand heavy chain (FIG. 6). More specifically, peaks a and b from the 0Lys recombinant antibody species treated with MGO were fractionated andanalyzed by reduced LC/MS. The top pane shows the corresponding lightchain mass spectra of the two peaks and the bottom pane depicts theheavy chain for the fractionated peaks. Mass heterogeneity of the chainscorresponding to +54 Da and +72 Da were observed for both fractions. Theresulting modifications are in agreement with the observations found inthe cell culture acidic peaks supporting the previous data that themodification is due to methylglyoxal. Thus, fractionation of theacidic-shifted 0 Lys material followed by LC/MS/MS tryptic mappingconfirmed that MGO modification of arginine residues was the cause ofthe observed adductions.

In addition, acid species from both cell culture and the MGO spike werecompared to each other by LC/MS/MS. The resulting MS/MS spectra showedfragmentation profiles that were highly comparable for mis-cleavages atarginine residues with the MGO adduction characteristic +54 Da and +72Da mass increases (FIG. 7). The data provide a confirmation that theacidic peaks resulting from the use of chemically defined media are dueto modifications of the expressed adalimumab recombinant antibody bymethylglyoxal which has accumulated in the cell culture bioreactor.Moreover, the modification of the arginine may influence thefragmentation of the peptide backbone. The strong similarities betweenthe two mass spectra further support the notion that the arginine hasundergone a modification which may result in destabilisation of thepeptide backbone.

Example 2 Functional Liabilities Associated with MethylglyoxalModifications to Adalimumab Antibodies

Methylglyoxal modifications of arginine residues lead to miscleavagesdue to the steric constraints imparted by the adducted MGO to the activesite of trypsin. In order to better quantitate and determine allsusceptible arginine residues in the adalimumab primary structure, anendoprotease Lys-C digestion was performed where arginine residues wereno longer recognized as target substrates in the peptide mappingprotocol. All Lys-C peptides were evaluated using the Sequest algorithmagainst the FASTA sequence for adalimumab. Several sites were identifiedas potential susceptible sites but one site of particular susceptibilitywas identified at R30 of the light chain. The sequences of the lightchain and heavy chain of the Adalimumab D2E7 are designated as SEQ IDNo. 1 and SEQ ID No. 2, respectively. A list of all potentialsusceptible arginine residues is shown in Table 1. Different sites mayhave different level of susceptibility to MGO modification. Not allsites have to be modified by MGO in a single molecule. Table 2 listspeptide fragments on Adalimumab that are susceptible to modification bymethylglyoxal.

TABLE 1 Potential Sites of MGO modification in Adalimumab AdalimumabLight Chain Adalimumab Heavy Chain Ab Chain Type (SEQ ID No. 1) (SEQ IDNo. 2) Arginine Sites Arginine 30 Arginine 16 Arginine 93 Arginine 259Arginine 108 Arginine 359 Arginine 420

TABLE 2 List of peptides susceptible to modification by methylglyoxalActivation RT NB Sequence Type Modifications Charge m/z [Da] MH+ [Da][min] Order EPQVYTLPPSrDELTK HCD R11(MGO (R) 72) 2 972.9988 1944.9927.71 MS2 EPQVYTLPPSrDELTK CID R11(MGO (R) 72) 3 649.0014 1944.99 27.72MS2 EPQVYTLPPSrDELTK CID R11(MGO) 3 642.9988 1926.982 27.81 MS2EPQVYTLPPSrDELTK HCD R11(MGO) 3 642.9988 1926.982 27.82 MS2EPQVYTLPPSrDELTK CID R11(MGO) 2 963.9942 1926.981 27.88 MS2EPQVYTLPPSrDELTK HCD R11(MGO) 2 963.9942 1926.981 27.89 MS2EVQLVESGGGLVQPGrSLR CID R16(MGO (R) 72) 2 1027.055 2053.103 32 MS2EVQLVESGGGLVQPGrSLR HCD R16(MGO (R) 72) 2 1027.055 2053.103 32.01 MS2EVQLVESGGGLVQPGrSLR CID R16(MGO) 3 679.0353 2035.091 32.11 MS2EVQLVESGGGLVQPGrSLR CID R16(MGO) 2 1018.05 2035.092 32.13 MS2EVQLVESGGGLVQPGrSLR HCD R16(MGO) 2 1018.05 2035.092 32.15 MS2DIQMTQSPSSLSASVGDrVTITcR HCD R18(MGO), 3 888.7587 2664.261 35.6 MS2C23(Carboxymethyl) DIQMTQSPSSLSASVGDrVTITcR HCD R18(MGO), 3 888.75832664.26 36.63 MS2 C23(Carboxymethyl) YNrAPYTFGQGTK CID R3(MGO (R) 72) 2787.8835 1574.76 17.61 MS2 YNrAPYTFGQGTK HCD R3(MGO (R) 72) 2 787.88351574.76 17.62 MS2 YNrAPYTFGQGTK CID R3(MGO (R) 72) 3 525.5911 1574.75917.63 MS2 YNrAPYTFGQGTK HCD R3(MGO (R) 72) 3 525.5911 1574.759 17.64 MS2YNrAPYTFGQGTKVEIK CID R3(MGO (R) 72) 2 1022.461 2043.916 46.16 MS2SLrLScAASGFTFDDYAMHWVR CID R3(MGO (R) 72), 3 888.4062 2663.204 49.36 MS2C6(Carboxymethyl) SLrLScAASGFTFDDYAMHWVR HCD R3(MGO (R) 72), 3 888.40622663.204 49.38 MS2 C6(Carboxymethyl) YNrAPYTFGQGTK CID R3(MGO) 2778.8782 1556.749 17.49 MS2 YNrAPYTFGQGTK HCD R3(MGO) 2 778.87821556.749 17.5 MS2 YNrAPYTFGQGTK CID R3(MGO) 3 519.5878 1556.749 17.56MS2 YNrAPYTFGQGTK HCD R3(MGO) 3 519.5878 1556.749 17.57 MS2 SFNrGEc HCDR4(MGO), 2 462.8614 924.7156 5.29 MS2 C7(Carboxymethyl)ASQGIrNYLAWYQQKPGK CID R6(MGO (R) 72) 3 727.3791 2180.123 32.15 MS2ASQGIrNYLAWYQQKPGK HCD R6(MGO (R) 72) 3 727.3791 2180.123 32.16 MS2ASQGIrNYLAWYQQKPGK CID R6(MGO (R)72) 2 1090.566 2180.125 32.2 MS2ASQGIrNYLAWYQQKPGK HCD R6(MGO (R)72) 2 1090.566 2180.125 32.21 MS2ASQGIrNYLAWYQQKPGK CID R6(MGO) 3 721.3756 2162.112 31.52 MS2ASQGIrNYLAWYQQKPGK HCD R6(MGO) 3 721.3756 2162.112 31.53 MS2ASQGIrNYLAWYQQKPGK CID R6(MGO) 2 1081.561 2162.115 31.55 MS2ASQGIrNYLAWYQQKPGK HCD R6(MGO) 2 1081.561 2162.115 31.56 MS2DTLMISrTPEVTcVVVDVSHEDPEVK CID R7(MGO (R)72), 1010.155 3028.451 44.42MS2 C13(Carboxymethyl) 3 DTLMISrTPEVTcVVVDVSHEDPEVK HCD R7(MGO (R)72),1010.155 3028.451 44.43 MS2 C13(Carboxymethyl) 3DTLMISrTPEVTcVVVDVSHEDPEVK CID R7(MGO), 1004.152 3010.442 44.14 MS2C13(Carboxymethyl) 3 DTLMISrTPEVTcVVVDVSHEDPEVK HCD R7(MGO), 1004.1523010.442 44.15 MS2 C13(Carboxymethyl) 3

The crystal structure of the adalimumab Fab unit in complex with itscognate binding partner TNF-alpha shows that R30 is intimately involvedin the contact surface between CDR1 and the antigen surface (FIG. 8).The figure shows the side chain of arginine 30 (indicated by arrow)protruding into the TNF-alpha structure (indicated by arrow). Amodification of this side chain by MGO would result in the formation ofa five-member ring originating at the guanidinium terminal of the sidechain and further penetrating into the TNF-alpha structure. The MGOmodification is therefore likely to impede adalimumab's ability to bindTNF-alpha due to steric constraints.

In order to further elucidate any functional liabilities associated withadalimumab and chemical modifications due to an accumulation of MGO in acell culture expression using chemically defined media, an enrichedMGO-modified fraction was isolated using weak cation exchangechromatography. A control fraction of a pure 0 Lys fraction was alsoobtained. The two fraction were analyzed by surface plasmon resonance tocalculate the association and dissociation rates of TNF-alpha to theimmobilized antibody. A three-fold reduction was observed for the MGOmodified adalimumab as compared to the 0 Lys control (FIG. 9). Thus, itappears that the methylglyoxal modification of Arginine 30 (R30) of thelight chain does impart a functional liability to the affectedpopulation of adalimumab drug substance. These data support thehypothesis that a chemical modification on the side chain of Arginine 30of the light chain induces steric interference with the CDR1 and theTNF-alpha binding surface which may lead to a significant drop inadalimumab potency. It is therefore desirable to reduce the amount ofthis modified form of antibody in adalimumab drug substance or drugproduct.

Example 3 Removal of Methylglyoxal-modified Adalimumab Using an AEXand/or CEX Strategy

A chromatographic strategy was employed to remove the early elutingacidic region on the WCX-10 chromatogram. After the removal process isperformed, adalimumab drug substance devoid of this region wasgenerated. As disclosed herein, expression of adalimumab in chemicallydefined media may cause an increase of species eluting in this acidicregion as a result of the accumulating MGO adducting to the positivelycharged guanidinium groups of the affected arginine residues. Thedisclosed chromatographic strategy helps clear this functional liabilityof the adalimumab preparation. The resulting adalimumab BDS is free ofor substantially free of the negative impact from the methylglyoxalmodification and has normal binding to its target, TNF-alpha.

The decision whether to use cationic exchange chromatography (CEX),anionic exchange chromatography (AEX), or both, to purify a protein isprimarily based on the overall charge of the protein. Therefore, it iswithin the scope of this invention to employ an anionic exchange stepprior to the use of a cationic exchange step, or a cationic exchangestep prior to the use of an anionic exchange step. Furthermore, it iswithin the scope of this invention to employ only a cationic exchangestep, only an anionic exchange step, or any serial combination of thetwo.

In performing the separation, the initial protein mixture can becontacted with the ion exchange material by using any of a variety oftechniques, e.g., using a batch purification technique or achromatographic technique.

For example, ion exchange chromatography is used as a purificationtechnique to separate the MGO-modified forms from the non-MGO-modifiedforms. Ion exchange chromatography separates molecules based ondifferences between the overall charge of the molecules. In the case ofan antibody, the antibody has a charge opposite to that of thefunctional group attached to the ion exchange material, e.g., resin, inorder to bind. For example, antibodies, which generally have an overallpositive charge in a buffer having a pH below its pI, will bind well tocation exchange material, which contain negatively charged functionalgroups.

In ion exchange chromatography, charged patches on the surface of thesolute are attracted by opposite charges attached to a chromatographymatrix, provided the ionic strength of the surrounding buffer is low.Elution is generally achieved by increasing the ionic strength (i.e.,conductivity) of the buffer to compete with the solute for the chargedsites of the ion exchange matrix. Changing the pH and thereby alteringthe charge of the solute is another way to achieve elution of thesolute. The change in conductivity and/or pH may be gradual (gradientelution) or stepwise.

Example 3.1 Removal of Methylglyoxal-Modified Adalimumab Using AEX

A process is described here for purifying a target protein product fromboth process and product related impurities. Specifically, a method isprovided for reducing product related charge variants (i.e. acidic andbasic species). The method involves contacting the process mixture withan anion exchange (AEX) adsorbent in an aqueous salt solution underloading conditions that permit both the target and non-target proteinsto bind to the AEX adsorbent and allowing the excess target molecule topass through the column and subsequently recovering the bound targetprotein with a wash at the same aqueous salt solution used in theequilibration (i.e. pre-loading) condition.

Source Material—

The antibody used in this study was derived from cell culture conditionsemploying both chemically defined media (CDM) and hydrolysate media. Theantibody was captured from the clarified harvest through affinitychromatography (Protein-A, GE MabSuRe) where the eluate is in a buffersystem of about 20 mM acetic acid at a pH of about 4.2.

Induced pH Gradient Anion Exchange Chromatography—

POROS 50PI (Applied Biosystems) resin was packed in 1.0 cm×10.0 cm(OmniFit) column. The column was equilibrated in a two-component buffercontaining acetate as the anion and either tromethalmine (Tris) orarginine as the cation. In these experiments, the anion (i.e. acetate)concentration was held constant and the cation (Tris/Arginine) was addedto achieve the desired pH. Induced pH gradients were initiallyperformed, without protein, by equilibrating the column with anAcetate/Tris or Acetate/Arginine buffer at pH 9.0 followed by a stepchange of the equivalent buffer at pH 7.0. Induced pH gradients withoutprotein were run at controlled acetate concentrations of 5 mM, 10 mM, 20mM, and 30 mM.

The POROS 50PI column was then loaded with 20 g/L of D2E7 in 5 mMAcetate/Tris (or Arginine) pH 9.0, followed by a 10 column volume (CV)isocratic wash, and then an induced pH gradient elution with a stepchange in the running buffer to 5 mM Acetate/Tris (or Arginine) pH 7.0.The column was then regenerated (5 CVs of 100 mM acetate+1 M NaCl),cleaned in place (3 CVs 1M NaOH, 60 min hold), and stored (5 CVs 20%ethanol). During elution, the column effluent was fractionated into0.5×CV and analyzed for UV280, WCX-10, and SEC (described below). TheD2E7 AEX-load was prepared by diluting the source material describedabove with Milli-Q water to 5 mM acetate and titrating with arginine tothe desired pH.

Flow-Through Anion Exchange Chromatography—

Using the induced pH gradient results, an operational pH was selected tooperate the POROS 50PI column in flow-through mode. The pH was selected(e.g. pH 8.8) to optimize the resolution between the acidic species andLysine variants. The first run was performed by loading 150 g/L in a 5mM Acetate/Arginine pH 8.8 buffer system, followed with a 20 CVisocratic wash. A FTW fraction was collected from 50-150 mAU andanalyzed for UV280, WCX-10, and SEC. The results from this run are shownin Table 3. This run was able to reduce acidic species by 60% and removealmost all detectable high molecular weight species (i.e. aggregates)with about 68% recovery.

TABLE 3 Acidic species and aggregates reduction by AEX AEX Poros 50PI,150 g/L FT, 5 mM Acetate/ Acidic Species SEC Arginine pH 8.8 AR1 + 2LysSum HMW Mono LMW AEX Load 17.805 81.685 1.704 97.947 0.348 (t = 0)AEX Load 19.711 79.746 1.975 97.831 0.194 (t = 10 days, 4° C.) AEX FTW7.085 92.108 0.019 99.889 0.092 (t = 0) AEX FTW 8.069 91.773 0.04 99.8530.107 (t = 10 days, 4° C.)

The data presented here demonstrates a method for the fine purificationof D2E7 from both product related (i.e. charge variants and molecularweight variants) impurities by loading the process stream to an anionexchange adsorbent under aqueous salt conditions (i.e. low conductivityand high pH) that permit both the target and non-target proteins to bindto the AEX adsorbent and allowing the excess target molecule to passthrough the column and subsequently recovering the bound target proteinwith a wash at the same aqueous salt solution used in the equilibration(i.e. pre-loading) condition.

Example 3.2 Removal of Methylglyoxal-Modified Adalimumab Using CEX

This Example describes a process for purifying a target protein productfrom both process and product related impurities by using a cationexchange (CEX) technique. Specifically, a reversible binding method isdisclosed for reducing product related charge variants (i.e. acidicspecies) of the target molecule. By way of example, the method mayinvolve some or all of the following steps.

In one step, the process mixture is caused to be in contact with acation exchange (CEX) adsorbent in an controlled aqueous buffer solutionwith pH and conductivity under loading conditions that permit both thetarget and non-target proteins to bind to the CEX adsorbent. The pH ofthe loading buffer is below the pI of the antibody molecule.

In another step, the charged variants, molecular variants and impuritiesare washed off using the same buffer conditions as the loading buffer.The product may then be eluted with a buffer having higher conductivitythan that of the loading buffer.

In this Example, three antibody molecules were used. Adalimumab antibodywas obtained from concentrated fractogel eluate in AY04 manufacturingprocess and CDM 300 L scale up run Protein A eluate. They were bufferexchanged into 29 mM Tris-acetate buffer pH 7.5 as CEX loading material.

Poros XS, (Applied Biosystems) strong CEX resin, CM Hyper D (Pall), weakCEX resin, Nuvia S (Bio-Rad) strong resin and GigaCap S 650 (TosohBiosciences) strong resin were packed in 1.0 cm×10.0 cm (OmniFit)columns. The column was equilibrated in a buffer system with appropriatepH and conductivity. The column load was prepared in the equilibrationbuffer and loaded on the column at 40 g protein/L resin followed bywashing with the equilibration buffer for 20 CV. The antibody productwas eluted out with 150 mM sodium chloride and 30 mM Tris-acetate buffersolution. 1M of NaCl was used for column regeneration and 1M of NaOHsolution was used for column cleaning.

Four buffer/salt systems, sodium chloride/Tris-acetate, Tris-acetate,Ammonium sulfate/Tris-acetate and arginine/Tris-acetate at different pHand conductivity were evaluated. The buffer conditions are listed inTable 4.

TABLE 4 Buffer conditions Resin Buffer pH Conductivity Poros XSTris-acetate 7.5, 6.5, 5.5 3 conductivity (strong) for each pH Sodiumchloride 7.5, 6.5, 5.5 3 conductivity for each pH Ammonium 7.5 3conductivity sulfate for each pH CM Hyper D Tris-acetate 7.5 3conductivity (weak) Sodium chloride 7.5, 6.8, 6.0 3 conductivity foreach pH Ammonium 7.5 3 conductivity sulfate Nuvia S Tris-acetate 7.5 3conductivity (strong) Sodium chloride 3 conductivity Ammonium 3conductivity sulfate GigaCap Tris-acetate 7.5 3 conductivity S 650

A reversible binding mode was performed using Adalimumab withTris-acetate buffer system. The loading utilised buffer at pH 7.5 andTris concentration at 145 mM with 40 g protein /L resin. The column washwas fractionated. The wash fractions and elute pool were analyzed byUV280, WCX-10 and SEC assays. The chromatogram is shown in FIG. 11.

Example 4 Charge Variants Reduction in Adalimumab by Poros XS Resin

In this Example, different resins and buffer conditions were evaluated.The starting material contained 14% total AR and 3% AR1. Experimentswere performed by varying resins and buffer conditions for acidicspecies removal. The results are described in the following sections.

Experiments were performed on Poros XS resin using NaCl to vary theconductivity with a fixed 29 mM Tris-acetate buffer for pH control.Three pH levels were tested, pH 7.5, 6.8 and 6.0. Each pH was studied atconductivities wherein the amount of NaCl was varied. As shown in FIG.12, acidic species can be removed by 3% with 90% yield. For furtherreduction in acidic species, the yields achieved vary under differentbuffer conditions. At pH 7.5 and 45 mM NaCl, the amount of acidicspecies was reduced by 6.8%, with 75% yield of Adalimumab. AR1 wassignificantly reduced to about zero percent, with a yield of 72% ofAdalimumab, and to less than 0.5% with over 80% yield of Adalimumab, asshown in Table 5. The column wash was fractionated and specified asFraction 1 to Fraction 5 by the order of adjacent to the eluate. TheAR1, AR2, Lys sum versus yield was calculated based on the results ofeach fraction.

TABLE 5 AR1 removal versus yield by CEX % % % Lys Yield Wash fractionsAR1 AR2 Sum (%) Load 2.9 12.1 84.3 n/a Eluate 0 7.8 92.2 72 Eluate +Fraction 1 0.3 8.8 91.0 79 Eluate + Fraction 1 + Fraction 2 0.6 9.6 89.883 Eluate + Fraction 1 + Fraction 2 + 1.6 10 88.4 88 Fraction 3 Eluate +Fraction 1 + Fraction 2 + 2.2 10.9 86.8 92 Fraction 3 +_Fraction 4Eluate + Fraction 1 + Fraction 2 + 2.9 11 86.1 93 Fraction 3 +_Fraction4 + Fraction 5

In summary, methods for the purification of Adalimumab from productrelated impurities (i.e. charge variants and molecular weight variants)are disclosed. More particularly, the process stream may be loaded to acation exchange adsorbent under appropriate aqueous conditions, whereinthe pH and conductivity of the loading and wash buffer is below the pIof the target protein that permit both the target protein and impuritiesto bind to the CEX adsorbent. The acidic species and other impuritiesmay then be washed off by using wash buffer which is the same as theloading buffer. Lastly, the bound target protein may be recovered byusing a high conductivity aqueous solution.

The present disclosure is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of this disclosure and the claims.

REFERENCES

The contents of all cited references (including literature references,patents, patent applications, and websites) that may be cited throughoutthis application or listed below are hereby expressly incorporated byreference in their entirety for any purpose into the present disclosure.The disclosure may employ, unless otherwise indicated, conventionaltechniques of immunology, molecular biology and cell biology, which arewell known in the art.

The present disclosure also incorporates by reference in their entiretytechniques well known in the field of molecular biology and drugdelivery. These techniques include, but are not limited to, techniquesdescribed in the following publications:

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EQUIVALENTS

The disclosure may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting of the disclosure. Scope of the disclosure is thusindicated by the appended claims rather than by the foregoingdescription, and all changes that come within the meaning and range ofequivalency of the claims are therefore intended to be embraced herein.

We claim:
 1. A composition comprising a binding protein capable ofbinding TNF-alpha, said binding protein comprising a methylglyoxal(MGO)-susceptible amino acid, wherein said composition is prepared bysubstantially removing molecules of said binding protein that compriseat least one MGO-modified amino acid.
 2. The composition of claim 1,wherein more than 70% of said molecules that comprise at least oneMGO-modified amino acid is removed.
 3. The composition of claim 1,wherein more than 90% of said molecules that comprise at least oneMGO-modified amino acid is removed.
 4. The composition of claim 1,wherein the MGO-susceptible amino acid is an arginine.
 5. Thecomposition of claim 1, wherein the binding protein is a human antibodyor an antigen-binding portion thereof, wherein the binding proteindissociates from human TNF-alpha with a K_(d) of 1×10⁻⁸ M or less and aK_(off) rate constant of 1×10⁻³ s⁻¹ or less, both determined by surfaceplasmon resonance, and wherein the binding protein neutralizes humanTNF-alpha cytotoxicity in a standard in vitro L929 assay with an IC₅₀ of1×10⁻⁷ M or less.